Abstract
The effect of structural dynamics on enzyme activity and thermostability has thus far only been investigated in detail for the serine protease alpha-chymotrypsin (for a recent review see Solá et al., Cell Mol Life Sci 2007, 64(16): 2133-2152). Herein, we extend this type of study to a structurally unrelated serine protease, specifically, subtilisin Carlsberg. The protease was incrementally glycosylated with chemically activated lactose to obtain various subtilisin glycoconjugates which were biophysically characterized. Near UV-CD spectroscopy revealed that the tertiary structure was unaffected by the glycosylation procedure. H/D exchange FT-IR spectroscopy was performed to assess the changes in structural dynamics of the enzyme. It was found that increasing the level of glycosylation caused a linearly dependent reduction in structural dynamics. This led to an increase in thermostability and a decrease in the catalytic turnover rate for both, the enzyme acylation and deacylation steps. These results highlight the possibility that a structural dynamics-activity relationship might be a phenomenon generally found in serine proteases.